Agrobacterium tumefaciens uses DNA transmission in pathogenesis. The transfer of part of its genetic material to plants results in the formation of tumors. Novel DNA transfer from bacterial cells to cells of a higher organism is an unique, interesting and important phenomenon in microbiology and biotechnology. Genes required for this promiscuous DNA transmission are organized in a 28.6 kb regulon located on a 200 kb Ti plasmid that also contains the 25 kb transferred DNA (T-DNA). The regulon is comprised of seven operons (vfrA, vfrB, vfrG, virC, vfrD, vfrH and vfrE), of which the virB operon is the largest containing eleven genes; whose protein products are primarily associated with the A. tumefaciens membrane, are required for virulence, but whose precise functions remain unknown. From our recent studies, the VirB proteins show striking similarities to Tra proteins of conjugative plasmids such as IncW plasmid R388 and IncF plasmid F. These Tra proteins are involved in the synthesis and assembly of the conjugative sex pilus. Thus, one part of this three part research program focuses on the VirB proteins, which will include analysis for pilin or pilin-like structures, molecular escorting by the VirB4 protein as well as other VirB proteins for identifying their topology on the bacterial membranes. The second part is on the negative regulation of vir genes by the Ros protein encoded by the ros chromosomal gene, which includes further analysis of the putative zinc finger in the Ros protein, the identification of specific residues involved in binding to the ros box, the binding of VirG to the vir box upstream of ros, and further defining the ros box in binding Ros. The third part of the research will be on the nature of the suppression of oncogenicity by the Osa protein encoded by the osa gene on IncW plasmid pSa. The work will include determining function and identifying potential competition sites at the inner membrane, where Osa resides. The research will therefore employ a combination of genetic, immunochemical, biochemical as well as high resolution microscopical techniques to gain further insights on the T-DNA transfer apparatus, vir gene regulation and oncogenic suppression.